Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry

First Advisor

Jimmie C. Oxley

Abstract

The toxicity or pharmacodynamics of many of the nitrated explosives have been well documented. Trinitrotoluene (TNT) is known to cause liver toxicity while nitrate esters (nitroglycerine) are known vasodilators. One class of explosive that has been on the rise due to the ease of manufacturing from household products are the peroxides. Of particular interest are the cyclic peroxides used for many home-made explosives (HME): triacetone triperoxide (TATP) and hexamethylene diamine triperoxide (HMTD). Very little is known about the toxicity or potentially beneficial effects of these compounds. This may be primarily due to the difficulty in detecting or working with these materials, particularly when they are extracted from living tissues. The use of liquid chromatography (LC) mass spectrometry (MS) is ideally suited to handle this type of sample, provided that the proper detection limits can be achieved. Additionally, this technique provides a very sensitive detection with gentle ionization for more definitive confirmation of the chemical in question over many other techniques historically chosen.

In our efforts to reduce the limits of quantification for TATP and HMTD, several remarkable discoveries were made. Most importantly, acetonitrile, one of the most commonly used LC/MS solvents used throughout many industries has shown direct inhibition of ionization. The proposed mechanism of this suppression is by the formation of neutral aggregates of the nitrile moiety with various, common functional groups. Peroxides are one of the most intensely affected moieties. Also, TATP and methyl ethyl ketone peroxide (MEKP) have been shown to react with one or more alcohols under atmospheric pressure ionization (API) conditions to produce new species which may be exploited to improve limits of detection. Caution must be used while working with these products since the conditions can directly affect the signal intensity and multiple related analytes can all provide this common product. Lastly, HMTD has been found to react with both primary and secondary amines and alcohols in the gas phase to produce unique products related to the nature of the amine or alcohol. This research has allowed limits of detection to improve by 20 to 50 times our original analysis limits.

Toxicity of HMTD and TATP were primarily in question. However, with the volatility associated with TATP, it seems prudent that this should be the first compound studied since the exposure to this chemical entity is highly probable for any scientist or animal (bomb-sniffing canines) working with it. Simple in vitro analysis using canine liver microsomes (DLM) and lung microsomes (DLgM) in the presence of NADPH (electron donor) were performed to determine the rate, product and nature of the metabolism. Since most of the Phase I metabolism associated with the cytochrome P450 (CYP) enzymes requires molecular oxygen, the incubations are performed in open containers. The exceptional volatility of solid TATP extended to solutions of the compound as well, thus preventing this technique for experimentation. To overcome this issue, oxygen gas was bubbled through the buffered solution used as the matrix for the in vitro studies prior to sealing the containers for the duration of the experiment. Based on this work, several discoveries have been made. The metabolism that does occur appears to be NADPH-dependent, which limits the types of enzymes with may be responsible. The affinity for the non-specific metabolism is very high, with a Km value of 2.21 μM (±14.8%) with a Vmax of 1.13 nmol/min/mg protein (±3.27%). This also indicates that the enzyme responsible for the metabolism is saturated at relatively low concentrations. Work with recombinant isoforms of specific CYP enzymes (rCYP) has shown that only rCYP2B11 has any effect (of the 5 major liver 5CYP’s commercially available) and that this metabolism is enhanced by the presence of cytochrome b5. The metabolism of CYP2B11 does not seem to account for all of the total metabolism of TATP.

Only one metabolite has been identified, the mono-oxidation of a single primary methyl carbon (TATP-OH), for TATP. Monitoring the relative amount of this metabolite has been performed. After the rate of metabolism of TATP begins to level, the TATP-OH begins to drop, without detection of a second metabolite. Attempts to trap a second metabolite with semicarbazide (for aldehydes and ketones) or glutathione (for soft electrophiles) did not provide any conclusive products related to the metabolism of these species. With the successful synthesis of TATP-OH, we were able to directly incubate this metabolite. Although it was metabolized more rapidly that TATP, we were unable to detect any metabolites. It was also shown to degrade to acetone in oxidized aqueous buffer, but this did not appear to be related to the metabolism. TATP metabolism was not affected by the presence of TATP-OH or additional undetected metabolite(s). TATP-OH is metabolized only by rCYP2B11, providing evidence that TATP and TATP-OH competitively compete for the same enzyme and TATP dominates this competition. Of particular note is that very little metabolism was observed with the lung microsomes compared to liver. This may have the consequence of significant systemic exposure to those coming into contact with this material.

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